Persistent Ascaris Transmission Is Possible in Urban Areas Even Where Sanitation Coverage Is High

In low-income, urban, informal communities lacking sewerage and solid waste services, onsite sanitation (sludges, aqueous effluent) and child feces are potential sources of human fecal contamination in living environments. Working in informal communities of urban Maputo, Mozambique, we developed a quantitative, stochastic, mass-balance approach to evaluate plausible scenarios of localized contamination that could explain why the soil-transmitted helminth Ascaris remains endemic despite nearly universal coverage of latrines that sequester most fecal wastes. We used microscopy to enumerate presumptively viable Ascaris ova in feces, fecal sludges, and soils from compounds (i.e., household clusters) and then constructed a steady-state mass-balance model to evaluate possible contamination scenarios capable of explaining observed ova counts in soils. Observed Ascaris counts (mean = −0.01 log10 ova per wet gram of soil, sd = 0.71 log10) could be explained by deposits of 1.9 grams per day (10th percentile 0.04 grams, 90th percentile 84 grams) of child feces on average, rare fecal sludge contamination events that transport 17 kg every three years (10th percentile 1.0 kg, 90th percentile 260 kg), or a daily discharge of 2.7 kg aqueous effluent from an onsite system (10th percentile 0.09 kg, 90th percentile 82 kg). Results suggest that even limited intermittent flows of fecal wastes in this setting can result in a steady-state density of Ascaris ova in soils capable of sustaining transmission, given the high prevalence of Ascaris shedding by children (prevalence = 25%; mean = 3.7 log10 per wet gram, sd = 1.1 log10), the high Ascaris ova counts in fecal sludges (prevalence = 88%; mean = 1.8 log10 per wet gram, sd = 0.95 log10), and the extended persistence and viability of Ascaris ova in soils. Even near-universal coverage of onsite sanitation may allow for sustained transmission of Ascaris under these conditions.

1. Text S1. Model framing 2. Text S2. Mass of soil containing Ascaris 3. Text S3. Recovery Experiments 4. Text S4. Microscopy training 5. Text S5. Soil Protocol 6. Figure S1. MapSan Trial Area 7. Figure S2. Empirical and simulated ova in soil 8. Text S6. Fecal Sludge Microscopy Protocol 9. Text S7. Presumptive Ascaris Viability Assessment 10. Figure S3. Equation for Decay Constant k 11. Figure S4. Empirical and simulated ova in fecal sludges 12. Figure S5. Empirical and simulated ova in stool 13. Table S1. Model parameters 14. Table S2. Comparison of ova counts in soil 15. Figure S6. Ascaris concentration by matrix 16. Figure S7. Ova in the system over time 17. Table S3. Sensitivity analysis 18. Figure S8. Chicken or ducks present 19. Figure S9. Dogs or cats present S2 Text S1. Model framing We used a mechanistic model to evaluate potential transport scenarios of Ascaris ova from child feces and fecal sludges to soils. There is evidence for household clustering of Ascaris infection. 1 We chose, however, to model a single hypothetical compound, instead of separate models from compounds with and without children shedding Ascaris ova for a number of reasons. First, we did not expect any difference in the quantity of child feces transported to soil at compounds with and without children infected by Ascaris. Next, we previously found that 88% of fecal sludge samples from MapSan trial tested positive Ascaris lumbricoides DNA, which suggests that other individuals besides the children who were enrolled in the trial may have been infected by Ascaris and would have been shedding Ascaris ova into the onsite sanitation system. Finally, there was a 1-13 month gap between when stool samples were collected (04/2017-04/2018) and when the soil samples were collected (05/2018). Given this gap, and the observed temporal heterogeneity in Ascaris infection (see tables below), we limited our approach to a single mechanistic model and did not attempt to account for household clustering in the model. Enumerators visited compounds enrolled in the MapSan trial and asked the compound leader to indicate the compound boundary, which was recorded using GeoODK (GeoMarvel, Alexandria, Virginia). From a random subset of these compound polygons (n=69), we used the measure tool in ArcMap 10.8.1 (ESRI, Redlands, California) to determine the surface area covered by the roofs of buildings, as a proxy for finished floors, because 94% (759/805) of enrolled households had a covered floor 2 . The median compound surface area ( ) was 124 square meters (min = 31, max = 530) and we estimated 74% of this living environment (min = 25%, max = 98%) was covered by housing or pavement ( ), resulting in a median soil surface area of 32 m 2 which may contain Ascaris ova. In addition, we assumed that ova were only transported to a depth of 0.5 cm (d) because evidence suggests soil-transmitted helminth ova are predominantly located at the surface 3 and there was a drought during the preceding three years 4 that limited the potential for ova to be transported into the subsurface 5 . Assuming the average density of soil (ρ ) is 1.7 g/cm 3 , we calculated the total mass of soil in the localized area was 274 kilograms (Equation 5) 6 .

Text S3. Recovery Experiments
We collected approximately 100 grams of sandy, silty, and loamy soil from the campus of the University of North Carolina at Chapel Hill in Chapel Hill, North Carolina. In a sterile 500 mL beaker we combined increments of 6-7 grams of each soil with 250 µL of fecal sludge containing 1,300 Ascaris lumbricoides ova per mL, pausing after each addition to homogenize the soil and fecal sludge for 30 seconds using a sterile wooden tongue depressor. This process resulted in a total mass of 25 grams of soil containing 1,300 Ascaris lumbricoides ova, which equates to 52 ova per gram wet soil. We placed the three soil samples in the fridge at 4 o C overnight. The following day we used the methods describe in Text S1 to enumerate the ova. We performed our methods in duplicate.

Limit of Detection Calculation
We analyzed approximately 4 grams of soil per sample, which indicates our theoretical limit of detection (LOD) was 0.25 ova/gram wet soil.  8 . Then, technicians were trained for one day on using a microscope and practiced identifying ova that were fixed and mounted onto pre-prepared slides (VWR, Radnor, PA).
The study team then acquired and trained on mini-FLOTAC with feces from dogs, cats, chickens, pigs, horses, and cows that contained a wide range of helminth ova (e.g., hookworm, Ascaris, Trichuris, Toxocara, strongyle, and pinworm) and artifacts (e.g., pollen, undigested food, fungal spores, and mite eggs). Frozen human stool samples collected as part of the MapSan Trial 2 that contained Ascaris ova were used to train technicians to differentiate between viable and non-viable Ascaris ova. As the final step in training, lab technicians had to demonstrate the ability to enumerate ova within 25% of the count observed by DC. During the analysis phase of the study, technicians consulted with DC and TB for help classifying ova if they were uncertain. After training as completed the study team intermittently analyzed animal feces with and without ova to continue training. These analyses served as microscopy process controls.  Ascaris k in soil at different temperatures S16 Figure S4. Empirical and simulated ova in fecal sludges S17 Figure S5. Empirical and simulated ova in stool S18  We conducted mixed-effects regression using the lme4 11 package in R. The exposure variable was compound location and the outcome variable was log10 transformed Ascaris ova concentration per wet gram soil. Non-detects were randomly imputed from 0.01 ova per gram to the LOD. Figure S6. Ascaris concentration by matrix Concentration of presumptively viable Ascaris ova by matrix (empirical data). The dashed red lines represent the limit of detection for each matrix. Non-detects were imputed up to 2 log10 below the LODs to visualize sample sizes. Fecal sludge samples were randomly selected from a set of samples that had tested positive for Ascaris lumbricoides via PCR.  Figure S7. Ova in the system over time

S21
This figure illustrates the total ova in the modeled compound soil if no child feces transport occurs for three years. The modeled fecal sludge emptying event would occur on the final day of the figure, bringing the quantity of ova back to the initial number of ova in the system on day 0.  Figure S8. Chicken or ducks present There was no difference in presumptively viable Ascaris ova concentrations in soils from compounds that had chickens or ducks present and those that did not. Figure S9. Dogs or cats present There was no difference in presumptively viable Ascaris ova concentrations in compounds that had dogs or cats present and those that did not.